The present invention relates to [[4-(2-azido-3-methyl-5-oxotetrahydrofuran-2-yl)phenyl]hydrazono]propanedinitrile (I) and its use as a reference compound in the analysis of batches of levosimendan synthesis, particularly in the determination of potentially genotoxic impurities in samples of a levosimendan batch. The present invention also relates to an analytic method for the determination of potentially genotoxic impurities in samples of a levosimendan batch wherein [[4-(2-azido-3-methyl-5-oxotetrahydrofuran-2-yl)phenyl]hydrazono]propanedinitrile (I) is used as a reference compound.
Levosimendan, which is the (xe2x88x92)-enantiomer of [[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]hydrazono]propanedinitrile, and the method for its preparation is described e.g. in EP 565546 B1. Levosimendan is potent in the treatment of heart failure and has significant calcium dependent binding to troponin. The use of levosimendan in the treatment of myocardial ischemia is described in WO 93/21921. The hemodynamic effects of levosimendan in man are described in Sundberg, S. et al., Am. J. Cardiol., 1995; 75: 1061-1066. Levosimendan is represented by the formula: 
Clinical studies have confirmed the beneficial effects of levosimendan in heart failure patients.
Levosimendan can be prepared in high purity and in nearly quantitative yields by treating (xe2x88x92)-6-(4-aminophenyl)-5-methyl-4,5dihydro-3(2H)pyridazinone with sodium nitrite and malononitrile as described in EP 565546 B1. However, it has been found that samples of levosimendan as synthesized show potential genotoxic properties occasionally in the bacterial mutagenity,test (Ames test).
It has now been found that the potential genotoxic properties of levosimendan samples are caused by an azido derivative impurity of formula (I) 
This azido derivative is a potent mutagen which can be formed during levosimendan synthesis in an amount sufficient to give a positive result in the bacterial mutagenity test. This compound named [[4-(2-azido-3-methyl-5-oxotetrahydrofuran-2-yl)phenyl]hydrazono]propanedinitrile (I) is therefore useful as a reference compound in the routine analysis of batches of levosimendan synthesis, particularly in the determination of potentially genotoxic impurities in levosimendan batches. The presence of (I) in a levosimendan batch indicates that further recrystallization is necessary for obtaining levosimendan material suitable for use as a medicine.